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Streptococcus pneumoniae is an important human pathogen in all age groups worldwide that causes a variety of diseases, ranging from life threatening septicaemia and meningitis to less severe sinusitis and otitis media. The factors that determine the virulence of S. pneumoniae are very complex but a key aspect of the organism's disease causing potential is the ability of the bacteria to regulate virulence factor expression and activity. In this study two main approaches were taken to investigate virulence gene expression in S. pneumoniae. Firstly, the feasibility of Recombinase based In vivo Expression Technology, RIVET, for use in S. pneumoniae to study gene expression in vitro, and then in vivo was assessed. However, the system was found to be unsuitable for use in this study. Secondly, the requirement for and the role of virulence gene regulators identified by Signature Tagged Mutagenesis were investigated. The requirement for different virulence gene regulators varied according to the murine model of infection used. Two of the regulators, MgrA and R1rA, were essential for nasopharyngeal carriage and production of pneumonia in mice by serotype 4 S. Pneumoniae. Both were shown to control the transcription of genes of a newly described pathogenicity islet, PPI2, encoding R1rA and proteins predicted to act at the bacterial cell surface. The PPI2 genes rlrA and rrgA were shown to be required for adhesion of serotype 4 S. pneumoniae to human epithelial cells and PPI2 gene expression was affected by the gaseous composition of the growth environment in an MgrA dependent manner. The distribution of MgrA, R1rA and PPI2 varied between clinical S. pneumoniae isolates emphasizing the likelihood of a different repertoire of virulence genes and regulators amongst different serotypes and strains of this important human pathogen.

Background: Streptococcus pneumoniae causes infections in children, older adults and the immunocompromised. This thesis investigates humoral immune responses to pneumococcal vaccination in asplenic adults, and responses to infection and vaccination in children with invasive pneumococcal disease (IPD). Methods: Pneumococal serotype-specific antibody was measured using IgG enzyme-linked immunosorbent assay (asplenic adults), multiplexed bead assays (lgG and IgA) and opsonophagocytic assay (children with IPD), and total IgG was measured using nephelometry. Results: Seven-valent pneumococcal conjugate vaccine (PCV7) elicited significant increases (p <0.01) in vaccine-serotype specific IgG from levels pre-to 3-6 weeks post-vaccination in III asplenic adults. Although 97% (108/111) previously had ~ 1 dose(s) of pneumococcal polysaccharide vaccine, compliance with other UK guidelines for prevention of post-splenectomy infection (meningococcal vaccines and antibiotic prophylaxis) was poor. PCV7 in children with IPD was immunogenic for vaccine serotypes, with IgG GMCs higher in the group immunised under the original licensed schedule than those under a reduced schedule for serotypes 4, 14, 19F and 23F (p <0.05). Thirteen children with age-appropriate vaccination, however, failed to respond to their infecting serotype following additional PCV7 vaccination; this immunological unresponsiveness persisted despite up to 2 further doses. This was the first reported observation of this phenomenon, which has since been seen in association with asymptomatic carriage of vaccine-serotype pneumococci. Conclusion: Although PCV7 was immunogenic in children with IPD and adults with asplenia, protection should not be assumed for individuals colonised by, or infected with, a vaccine serotype when vaccinated. Further work is required to investigate causes of immunological unresponsiveness.

Streptococcus pneumoniae naturally resides in the nasopharynx of colonised individuals. Although nasopharyngeal colonisation is asymptomatic it is the first step toward pneumococcal disease. Elucidating the mechanisms by which S. pneumoniae stimulates the mucosal innate immune response may help us understand the progression from asymptomatic colonisation to inflammatory disease.

Streptococcus pneumoniae is one of the major human pathogens and causes one to two million deaths every year as a result of meningitis, pneumoniae and sepsis. The pneumococcal cell wall consists of peptidoglycan and teichoic acids and plays an important role in virulence. The peptidoglycan maintains shape and osmotic stability of the cell. The synthesis of peptidoglycan is an important target for antibodies. The teichoic acids (TA) are differentiated in wall teichoic acids (WTA) and lipoteichoic acid (LTA). Both, LTA and WTA, consist of identical subunits, implying that their precursors originate from the same synthesis pathway. The pneumococcal TAs are decorated with choline, a compound that is very rare among bacteria. Choline is essential for the growth of S. pneumoniae. The choline residues bound to the TAs serve as anchors for the class of the choline binding proteins (CBPs), which includes important virulence factors such as LytA and PspA. The enzymes, which are responsible for the choline metabolism, are encoded in the lie region. In the last three decades numerous basic tools for cell biology research have been established for model organisms as E. coli. Many of these tools were not available for S. pneumoniae. In this work, novel tools for cell biology and cell wall analysis were established. The new cell biology methods include time-lapse microscopy of live cells and cellular localisation of GFP-tagged proteins, as well as a new Zn-r-Inducible promoter for efficient gene depletion. Using these tools, the gene products of the lie locus have been localised, providing the first evidence that the loading of WTA with phosphorylcholine residues is a membrane-associated process catalysed by the LicDl and LicD2 enzymes. The new cell wall methods include a protocol for the isolation of WTA chains and determination of the muropeptide pattern from isolated peptidoglycan. In this work, WTA chains were isolated for the first time and analysed by mass spectrometry. Additionally, the muropeptide profile of the laboratory strain R6 was established and new modifications of the pneumococcal peptidoglycan were found, which have not been described before. These novel tools will be very valuable to study the cell biology and the cell wall of the human pathogen S. pneumoniae.

The identification of four sortase homologues srtA-D in the pneumococcal genome prompted their investigation as candidate pneumococcal virulence factors. Not all of these sortase genes were present in both sequenced strains and so sortase gene distribution was investigated among a collection of clinical isolates. In contrast to srtB, C and D, srtA was found in all strains examined and so was selected for further study. It was subsequently found to contribute to pneumococcal virulence in mouse models of pneumonia, bacteraemia and colonisation. This is the first demonstration of a contribution of srtA to pneumococcal virulence. To complement this examination of srtA, some of the surface proteins known or likely to be anchored by SrtA were also investigated for a role in virulence in the animal model of pneumonia. In addition, two genes, annotated in the pneumococcal genomes as a macrophage infectivity potentiator protein and an exfoliative toxin A were also investigated and found to be novel pneumococcal virulence factors. However, it appears they have been incorrectly annotated and these genes do not represent a macrophage infectivity potentiator protein and exfoliative toxin A. Instead, one of them seems to be involved in the response to oxidative stress while no function for the other can yet be ascertained. Janus mutagenesis is a novel technique for manipulation of the pneumococcal genome allowing the creation of mutations that lack a selectable marker. This provides an accessible and potentially powerful method to easily alter the genome to make informative mutations. This thesis describes the first use, to our knowledge, of Janus mutagenesis to investigate pneumococcal virulence.

Infections due to Streptococcus pneumoniae (the pneumococcus) remain a substantial source of morbidity and mortality in both developing and developed countries despite a century of research and the development of effective therapeutic interventions (such as antibiotic therapy and vaccination). The ability of the pneumococcus to evade multiple classes of antibiotic through several genetically determined resistance mechanisms and its evasion of capsular polysaccharide based vaccines through serotype replacement and capsular switching, all reflect the extensive diversity and plasticity of the genome of this naturally transformable organism which can readily alter its genome in response to its environment and the pressures placed upon it in order to survive. The purpose of this thesis is to investigate this diversity from a genome sequence perspective and to relate these observations to pneumococcal molecular epidemiology in a region of high biodiversity, the pathogenesis of certain disease manifestations and assess for a possible bacterial genetic basis for the pneumococcal phenotypes of, “carriage” and, “invasion.” In order to do this, microarray comparative genomic hybridization (CGH) has been utilized to compare DNA from a variety of pneumococcal isolates chosen from 10 diverse serotypes and Multilocus Sequence Types and from clinically relevant serotypes and sequence types (particularly serotypes 3, 4 and 14 and sequence types ST9, ST246 and ST180)) against a reference, sequenced pneumococcal genome from an extensively investigated serotype 4 isolate – TIGR4. Microarray comparison of the transcriptional profiles of several isolates has also been undertaken to compare gene expression from isolates of serotype 1 (ST227 and ST306) and serotype 3 (ST180) related to particular disease states and exposure of a multi-resistant pneumococcus to an antimicrobial (clarithromycin) commonly used to treat pneumococcal pneumonia.

Department of Microbiology / PhD (Microbiology)

Streptococcus pneumoniae (the pneumococcus) is a bacterial pathogen responsible for >1.6 million annual deaths globally. Pneumococcal penicillin-resistance is conferred by acquisition of ‘altered’ penicillin-binding protein (pbp) genes. The first penicillin-nonsusceptible pneumococci were identified in the late 1960s. Global pneumococcal penicillin-nonsusceptibility rates rapidly increased in the 1980s/90s. Since 2000, protein-conjugate vaccines, targeting 7, 10 or 13 of the ≥94 different pneumococcal capsule types (serotypes), have been introduced in many countries. Following vaccine implementation there has been a decline in vaccine-type pneumococcal disease and an increase in non-vaccine-type disease. These epidemiological changes result from “serotype replacement” and/or “serotype switching”. The former describes the expansion of non-vaccine-type clones in the absence of vaccine-type pneumococci. The latter describes serotype change following recombination at the capsule polysaccharide synthesis (cps) locus. To fully understand how pneumococci respond to vaccine- and antibiotic-induced selective pressures, we must better understand the evolutionary history of this pathogen. This thesis describes the study of a global collection of 426 pneumococci, dated 1937 - 2007. Serotype, genotype and penicillin-susceptibility data were collected. Nucleotide sequences of three pbp genes (for 389 isolates) and whole-genome sequences (for 96 isolates) were also generated. The data demonstrated the long-term persistence of certain clones within pneumococcal populations, and that pbp and large-fragment (>30 kb) cps ± pbp recombination was occurring prior to both widespread antibiotic use and vaccine implementation. The data highlighted the promiscuous nature of the globally-distributed PMEN1 clone and its contribution to the spread of pneumococcal penicillin-resistance. PMEN1 also donated multiple, large regions (1.7 - 32.3 kb) of its genome to at least two un-related clones. Finally, six “Tn916-like” genetic elements, conferring resistance to non-penicillin antibiotics, were newly identified. These included two of the oldest ever described. These results provided a unique insight into the history of pneumococcal evolution and the importance of genetic recombination.